Conotoxin peptides

ABSTRACT

The present invention is directed to conotoxin peptides having 25-35 amino acids, six cysteines which form three disulfide bonds between the first and fourth, second and fifth, and third and sixth cysteines, respectively. The invention is directed to δ-conotoxin GmVIA having the formula Val-Lys-Pro-Cys-Arg-Lys-Glu-Gly-Gln-Leu-Cys-Asp-Pro-Ile-Phe-Gln-Asn-Cys-Cys-Arg-Gly-Trp-Asn-Cys-Val-Leu-Phe-Cys-Val (SEQ ID NO:1). This peptide activates sodium channels. The invention is further directed to μO-conotoxin peptides of the generic formula Ala-Cys-Xaa 1  -Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Xaa 2  -Gly-Phe-Xaa 3  -Tyr-Cys-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val, wherein Xaa 1  is Arg or Ser, Xaa 2  is Ile or Leu and Xaa 3  is Ile or Val (SEQ ID NO:2). These peptides block sodium channels. Examples of μO-conotoxin peptides of the present invention are MrVIA, having the formula Ala-Cys-Arg-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Ile-Gly-Phe-Ile-Tyr-Cys-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val (SEQ ID NO:3), and MrVIB, having the formula Ala-Cys-Ser-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Leu-Gly-Phe-Val-Tyr-Cys-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val (SEQ ID NO:4).

This invention was made with Government support under Grant Nos.GM-48677 and NS-27219 awarded by the National Institutes of Health,Bethesda, Md. The United States Government has certain rights in theinvention.

This is a continuation of application Ser. No. 08/319,554, filed Oct. 7,1994, U.S. Pat. No. 5,719,264 incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to relatively short peptides, and moreparticularly to peptides between about 25 and about 35 residues inlength, which are naturally available in minute amounts in the venom ofthe cone snails or analogous to the naturally available peptides, andwhich include three cyclizing disulfide linkages.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are numerically referenced in thefollowing text and respectively grouped in the appended bibliography.

Mollusks of the genus Conus produce a highly toxic venom which enablesthem to carry out their unique predatory lifestyle. Prey are immobilizedby the venom which is injected by means of a highly specialized venomapparatus, a disposable hollow tooth which functions both in the mannerof a harpoon and a hypodermic needle.

Few interactions between organisms are more striking than those betweena venomous animal and its envenomated victim. Venom may be used as aprimary weapon to capture prey or as a defense mechanism. These venomsdisrupt essential organ systems in the envenomated animal, and many ofthese venoms contain molecules directed to receptors and ion channels ofneuromuscular systems.

The predatory cone snails (Conus) have developed a unique biologicalstrategy. Their venom contains relatively small peptides that aretargeted to various neuromuscular receptors and may be equivalent intheir pharmacological diversity to the alkaloids of plants or secondarymetabolites of microorganisms. Many of these peptides are among thesmallest nucleic acid-encoded translation products having definedconformations, and as such, they are somewhat unusual. Peptides in thissize range normally equilibrate among many conformations. Proteinshaving a fixed conformation are generally much larger.

The cone snails that produce these toxic peptides, which are generallyreferred to as conotoxins or conotoxin peptides, are a large genus ofvenomous gastropods comprising approximately 500 species. All cone snailspecies are predators that inject venom to capture prey, and thespectrum of animals that the genus as a whole can envenomate is broad. Awide variety of hunting strategies are used; however, every Conusspecies uses fundamentally the same basic pattern of envenomation.

The major paralytic peptides in these fish-hunting cone venoms were thefirst to be identified and characterized. In C. geographus venom, threeclasses of disulfide-rich peptides were found: the α-conotoxin peptides(which target and block the nicotinic acetylcholine receptors); theμ-conotoxin peptides (which target and block the skeletal muscle Na⁺channels); and the ω-conotoxin peptides (which target and block thepresynaptic neuronal Ca²⁺ channels). However, there are multiplehomologs in each toxin class; for example, there are at least fivedifferent ω-conotoxin peptides present in C. geographus venom alone.Considerable variation in sequence is evident, and when differentω-conotoxin peptide sequences were first compared, only the cysteineresidues that are involved in disulfide bonding and one glycine residuewere found to be invariant. Another class of conotoxins found in C.geographus venom is that referred to as conantokins, which cause sleepin young mice and hyperactivity in older mice and are targeted to theNMDA receptor. Each cone venom appears to have its own distinctivegroup, or signature, of different conotoxin sequences.

Many of these peptides have now become fairly standard research tools inneuroscience and can be used as chemical probes for receptors and ionchannels (1). μ-Conotoxin peptides, because of their ability topreferentially block muscle but not axonal Na⁺ channels, are convenienttools for immobilizing skeletal muscle without affecting axonal orsynaptic events. ω-Conotoxin peptides have become standardpharmacological reagents for investigating voltage-sensitive Ca²⁺channels and are used to block presynaptic termini and neurotransmitterrelease. Several conotoxin peptides have also found utility in screeningnewly isolated conotoxin peptides or analogs for medical purposes (2).

SUMMARY OF THE INVENTION

The present invention is directed to conotoxin peptides having 25-35amino acids, six cysteines which form three disulfide bonds between thefirst and fourth, second and fifth, and third and sixth cysteines,respectively. The invention is directed to δ-conotoxin GmVIA having theformulaVal-Lys-Pro-Cys-Arg-Lys-Glu-Gly-Gln-Leu-Cys-Asp-Pro-Ile-Phe-Gln-Asn-Cys-Cys-Arg-Gly-Trp-Asn-Cys-Val-Leu-Phe-Cys-Val(SEQ ID NO:1). This peptide activates sodium channels. The invention isfurther directed to μO-conotoxin peptides of the generic formulaAla-Cys-Xaa₁ -Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Xaa₂ -Gly-Phe-Xaa₃-Tyr-Cys-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val, wherein Xaa₁is Arg or Ser, Xaa₂ is Ile or Leu and Xaa₃ is Ile or Val (SEQ ID NO:2).These latter peptides block sodium channels.

Examples of μO-conotoxin peptides of the present invention are MrVIA,having the formulaAla-Cys-Arg-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Ile-Gly-Phe-Ile-Tyr-Cys-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val(SEQ ID NO:3), and MrVIB, having the formulaAla-Cys-Ser-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Leu-Gly-Phe-Val-Tyr-Cys-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val(SEQ ID NO:4).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is directed to conotoxin peptides having 25-35amino acids, six cysteines which form three disulfide bonds between thefirst and fourth, second and fifth, and third and sixth cysteines,respectively. The invention is directed to δ-conotoxin GmVIA having theformulaVal-Lys-Pro-Cys-Arg-Lys-Glu-Gly-Gln-Leu-Cys-Asp-Pro-Ile-Phe-Gln-Asn-Cys-Cys-Arg-Gly-Trp-Asn-Cys-Val-Leu-Phe-Cys-Val(SEQ ID NO:1). This peptide activates sodium channels and is useful aspesticides, e.g. against garden snails and slugs, using conventionaltechniques, including sprinkling, spraying or creating transgenicplants. The invention is further directed to μO-conotoxin peptides ofthegenericformulaAla-Cys-Xaa₁-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Xaa₂ -Gly-Phe-Xaa₃-Tyr-Cys-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val, wherein Xaa₁is Arg or Ser, Xaa₂ is Ile or Leu and Xaa₃ is Ile or Val (SEQ ID NO:2).These latter peptides block sodium channels and are useful as activeagents for anti-seizures as are other sodium channel blockers.

Examples of μO-conotoxin peptides of the present invention are MrVIA,having the formulaAla-Cys-Arg-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Ile-Gly-Phe-Ile-Tyr-Cys-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val(SEQ ID NO:3), and MrVIB, having the formulaAla-Cys-Ser-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Pro-Ile-Leu-Gly-Phe-Val-Tyr-Cys-Cys-Pro-Gly-Leu-Ile-Cys-Gly-Pro-Phe-Val-Cys-Val(SEQ ID NO:4).

Despite the close relationship of the δ-conotoxins to the ω-conotoxins,it is clear that they have different physiological targets. Theω-conotoxins inhibit voltage-gated Ca²⁺ channels, distinguishing varioussubtypes. In contrast, the δ-conotoxins are without effect on Ca²⁺channels; the results shown below demonstrate that that they do notcompete for binding with ω-conotoxin GVIA and do not induce the shakingsyndrome in mice characteristic of the ω-conotoxins.

The electrophysiological results presented below demonstrate thatδ-conotoxin GmVIA prolongs spike duration by slowing the inactivationkinetics of the sodium current, and thus at the gross physiologicallevel it appears to have effects similar to those of δ-conotoxin TxVIA(3). Detailed electrophysiological studies described herein provideevidence that δ-conotoxin GmVIA specifically targets Na⁺ channels andprolongs the action potential duration by slowing down the sodiumcurrent inactivtion. The data clearly indicate that there aresignificant differences between GmVIA and TxVIA at a detailedmechanistic level. In many ways, this is not surprising because of thetremendous sequence divergence between GmVIA and TxVIA.

Biologically active δ-conotoxin GmVIA has been chemically synthesized,demonstrating that the biological activity is not due to contaminants. Adifferent family of Conus peptides, the μ-conotoxins, is known whichalso affects Na⁺ channels. However, these have a different disulfideframework, are channel blockers specific for the muscle subtype, and,like the ω-conotoxins, are highly basic molecules. Given the verydifferent chemical character of δ-conotoxins, it is likely that theirsite of action on the Na⁺ channel is quite distinct.

δ-Conotoxin GmVIA, the major component present in the venom of themollusc hunting snail C. gloriamaris, elicits spike broadening at aconcentration of 0.5 μM. Voltage clamp analysis reveals that the effectsof the toxin are expressed by: Slowing down the rate of the early sodiumcurrent inactivation; induction of a late slowly inactivating sodiumcurrent which is not detectable in control experiments; Shifting thesteady state sodium current inactivation curve to more depolarizedvalues and shifting the activation curve to more hyperpolarized values.These changes are not associated with an increase in the rate of rise ofthe early sodium current or by a change in the peak sodium current.

Sodium current inactivation experiments show that the toxin modifies thesodium current inactivation kinetics froma single exponential with anaverage τ=0.47±0.14 ms to a slower decay which can be described by twotime constants: the initial inactivation phase has a τ=0.86±0.12 ms, andthe second phase a τ=488±120 ms. These changes in time constants mayaccount for the typical alterations in the action potential shapeinduced by δ-conotoxin GmVIA. In artificial sea water (ASW) and in thepresence of the toxin, the early fast phase of the action potential andits plateau corresponds to the early rapidly inactivating sodium currentand the long-lasting, slowly inactivating phase.

The amplitude of the late phase of the sodium current induced byδ-conotoxin GmVIA is independent of the amplitude of the early current.Thus, even when the early current is completely inactivated by aprepulse, the amplitude of the late component is not reduced. Thesimplest explanation to account for this observation is that the toxinalters the inactivation kinetics of the entire population of sodiumchannels from a single time constant to dual time constants. If thisassumption is right, then the observations suggest that in the presenceof δ-conotoxin-GmVIA, the sodium channels can be inactivated in twomodes: In the one mode they go through the rapid inactivation phase tothe slow inactivating phase, or alternatively are "switched" directlyinto the slowly inactivating mode without going first through therapidly inactivating phase.. An alternative explanation assumes that thetoxin activates a population of silent, slowly inactivating sodiumchannels. This hypothesis is also consistent with the finding tht thenon-inactivating phase can be activated independently of the earlysodium current. However, since the peak of the early sodium current isnot increased in the presence of the toxin as would be expected ifadditional sodium channels were activated, then this hypothesis seems tobe less likely.

The macroscopic effects of the toxin purified from the venom of theshallow water molusc hunting snail C. textile (3) and the one purifiedfrom the deep water C. gloriamaris are similar but not identical. Bothtoxins alter the inactivation kinetics of the sodium current from aprocess that is best described by a single exponent into a biphasicprocess. However, the effects of the two toxins differ becauseδ-conotoxin GmVIA induces a significantly longer, slow inactivationphase than that induced by δ-conotoxin TxVIA. Additionally, the effectsof δ-conotoxin TxVIA undergo desensitization in the presence of thetoxin, whereas the effects of δ-conotoxin GmVIA are observed for as longas the toxin is present in the bathing solution. It is reasonable toassume that these peptide toxins target the same ligand binding pocketof the sodium channel: Both peptides are extremely hydrophobic, acharacteristic which may be important for binding to this specific site.Nevertheless, the differences in the primry sequences of these peptidescause a clear difference in their detailed effects on sodium currentinactivation kinetics.

Despite the close relationship of the precursor sequences of theμO-conotoxins to ω-conotoxin GVIA and to δ-conotoxin TxVIA, it is clearthat they have different physiological targets. The ω-conotoxins inhibitvoltage-gated Ca²⁺ channels, distinguishing various subtypes. Theδ-conotoxins activate Na⁺ channels. In contrast, the μO-conotoxins arewithout effect on Ca²⁺ channels; the results show that they do notcompete for binding with ω-conotoxin GVIA and do not induce the shakingsyndrome in mice characteristic of the ω-conotoxins. Instead ofactivating the Na⁺ channels, the μO-conotoxins block these channels.

The electrophysiological results presented below demonstrate thatμO-conotoxin MrVIA specifically targets Na⁺ channels and blocks theaction potential and inward sodium current. The block is not associatedwith a change in the current voltage relationships. Biologically activeμO-conotoxins have been chemically synthesized, demonstrating that theactivity is not due to contaminants.

Voltage clamp analysis reveals that ten seconds after toxin applicationto reach a final bath concentration of 350 nM, the sodium actionpotential was blocked. An increase in the stimulus intensity after theblockade of the action potential failed to elicit a regenerativeresponse. To directly examine the toxin action on sodium, calcium andpotassium currents, the whole-cell patch clamp configuration was used.The inward I_(Na+) evoked by depolarizing the neuron from a holdingpotential of -50 to 20 mV was completely blocked 30 seconds followingthe application of 250 nM MrVIA. Partial blockage of I_(Na+) by 40 nMtoxin revealed that the block is not associated with a change in thecurrent voltage relationships. Patch clamp experiments revealed thatcalcium and potassium currents are not affected by the toxin.

Binding competition experiments demonstrate that competitive bindinginhibition by μO-conotoxin MrVIA does not occur for the high affinityω-conotoxin GVIA binding site on mammalian brain Ca²⁺ channels.Electrophysiological experiments show that μO-conotoxin MrVIA andδ-conotoxin TxVIA elicit oposite effects, since δ-conotoxin TxVIA is anexcitotoxin which increases Na⁺ conductance (3). Thus, although μO-, ω-and δ-onotoxins apparently belong to the same protein superfamily, theyhave strickingly different physiological effects. In contrast, thefunctionally homologous μ-conotoxin GIIIA has an unrelated disulfidestructure, and its precursor sequence shows no homology whatsoever tothe μO-conotoxins from C. marmoreus. Thus, the peptides providemolecular guideposts for species diversification in this genus. Thegenetic analysis shows that the μO-conotoxins, Na channel inhibitorsfrom C. marmoreus, were independently evolved from the μ-conotoxins fromfish-hunting Conus. Thus, in this single genus, one protein superfamilycomprises multiple functionally-distinct toxin clases, but functionalconvergence of two sodium channel-blocking toxins from differentsuperfamilies is also observed.

These peptides, which are generally termed δ- or μO-conotoxin peptides,are sufficiently small to be chemically synthesized. General chemicalsyntheses for preparing the foregoing conotoxin peptides are describedhereinafter, along with specific chemical syntheses of several conotoxinpeptides and indications of biological activities of these syntheticproducts. Various ones of these conotoxin peptides can also be obtainedby isolation and purification from specific Conus species using thetechnique described in U.S. Pat. No. 4,447,356 (4), the disclosure ofwhich is incorporated herein by reference.

Although the conotoxin peptides of the present invention can be obtainedby purification from the enumerated cone snails, because the amounts ofconotoxin peptides obtainable from individual snails are very small, thedesired substantially pure conotoxin peptides are best practicallyobtained in commercially valuable amounts by chemical synthesis. Forexample, the yield from a single cone snail may be about 10 microgramsor less of conotoxin peptide. By "substantially pure" is meant that thepeptide is present in the substantial absence of other biologicalmolecules of the same type; it is preferably present in an amount of atleast about 85% by weight and preferably at least about 95% of suchbiological molecules of the same type which are present (i.e., water,buffers and innocuous small molecules may be present). Chemicalsynthesis of biologically active conotoxin peptides depends of courseupon correct determination of the amino acid sequence.

The conotoxin peptides can also be produced by recombinant DNAtechniques well known in the art. Such techniques are described bySambrook et al. (5) The peptides produced in this manner are isolated,reduced if necessary, and oxidized to form the correct disulfide bonds.

One method of forming disulfide bonds in the conotoxin peptides of thepresent invention is the air oxidation of the linear peptides forprolonged periods under cold room temperatures. This procedure resultsin the creation of a substantial amount of the bioactive,disulfide-linked peptides. The oxidized peptides are fractionated usingreverse-phase high performance liquid chromatography (HPLC) or the like,to separate peptides having different linked configurations. Thereafter,either by comparing these fractions with the elution of the nativematerial or by using a simple assay, the particular fraction having thecorrect linkage for maximum biological potency is easily determined. Itis also found that the linear peptide, or the oxidized product havingmore than one fraction, can sometimes be used for in vivo administrationbecause the cross-linking and/or rearrangement which occurs in vivo hasbeen found to create the biologically potent conotoxin molecule.However, because of the dilution resulting from the presence of otherfractions of less biopotency, a somewhat higher dosage may be required.

A second method of forming the disulfide bonds in the conotoxin peptidesof the present invention involves the use of acetamidomethyl (Acm) asprotection agent on the second and fifth cysteines during the synthesisof the conotoxin peptides. The use of Acm on these two residues is basedon the analogy with disulfide bridges in other conotoxin peptides. Thepeptide with the Acm protected cysteines is air-oxidized overnight atroom temperature. The bicyclic peptides are separated by highperformance liquid chromatography (HPLC) and the desired isomerisolated. The final disulfide bridge is carried out by iodination. Theundesired isomers are efficiently recycled by reduction to linearpeptide. The desired isomer is determined by a partial reductionanalysis (6). In this analysis, a sample of a bicyclic precursor istreated with tris-[2-carboxyethyl]-phosphine to give linear peptide anda singly-bridged intermediate. The latter peptide is reacted withiodoacetamide, and the location of alkylated cysteine residues isestablished by sequence analysis. In this analysis, it was determinedthat the correct linkages were between the first and fourth, second andfifth, and third and sixth cysteines for GmVIA, for example..

The peptides are synthesized by a suitable method, such as byexclusively solid-phase techniques, by partial solid-phase techniques,by fragment condensation or by classical solution couplings. Theemployment of recently developed recombinant DNA techniques may be usedto prepare these peptides, particularly the longer ones containing onlynatural amino acid residues which do not require post-translationalprocessing steps.

In conventional solution phase peptide synthesis, the peptide chain canbe prepared by a series of coupling reactions in which the constituentamino acids are added to the growing peptide chain in the desiredsequence. The use of various N-protecting groups, e.g.,dicyclohexylcarbodiimide or carbonyldimidazole, various active esters,e.g., esters of N-hydroxyphthalimide or N-hydroxysuccinimide, and thevarious cleavage reagents, to carry out reaction in solution, withsubsequent isolation and purification of intermediates, is well knownclassical peptide methodology. Classical solution synthesis is describedin detail in the treatise, "Methoden der Organischen Chemie(Houben-Weyl): Synthese von Peptiden," (7). Techniques of exclusivelysolid-phase synthesis are set forth in the textbook, "Solid-PhasePeptide Synthesis," (8), and are exemplified by the disclosure of U.S.Pat. No. 4,105,603 (9). The fragment condensation method of synthesis isexemplified in U.S. Pat. No. 3,972,859 (10). Other available synthesesare exemplified by U.S. Pat. Nos. 3,842,067 (11) and 3,862,925 (12).

Common to such chemical syntheses is the protection of the labile sidechain groups of the various amino acid moieties with suitable protectinggroups which will prevent a chemical reaction from occurring at thatsite until the group is ultimately removed. Usually also common is theprotection of an α-amino group on an amino acid or a fragment while thatentity reacts at the carboxyl group, followed by the selective removalof the α-amino protecting group to allow subsequent reaction to takeplace at that location. Accordingly, it is common that, as a step insuch a synthesis, an intermediate compound is produced which includeseach of the amino acid residues located in its desired sequence in thepeptide chain with appropriate side-chain protecting groups linked tovarious ones of the residues having labile side chains.

As far as the selection of a side chain amino protecting group isconcerned, generally one is chosen which is not removed duringdeprotection of the α-amino groups during the synthesis. However, forsome amino acids, e.g., His, protection is not generally necessary. Inselecting a particular side chain protecting group to be used in thesynthesis of the peptides, the following general rules are followed: (a)the protecting group preferably retains its protecting properties and isnot split off under coupling conditions, (b) the protecting group shouldbe stable under the reaction conditions selected for removing theα-amino protecting group at each step of the synthesis, and (c) the sidechin protecting group must be removable, upon the completion of thesynthesis containing the desired amino acid sequence, under reactionconditions that will not undesirably alter the peptide chain.

It should be possible to prepare many, or even all, of these peptidesusing recombinant DNA technology. However, when peptides are not soprepared, they are preferably prepared using the Merrifield solid-phasesynthesis, although other equivalent chemical syntheses known in the artcan also be used as previously mentioned. Solid-phase synthesis iscommenced from the C-terminus of the peptide by coupling a protectedα-amino acid to a suitable resin. Such a starting material can beprepared by attaching an α-amino-protected amino acid by an esterlinkage to a chloromethylated resin or a hydroxymethyl resin, or by anamide bond to a benzhydrylamine (BHA) resin or paramethylbenzhydrylamine(MBHA) resin. Preparation of the hydroxymethyl resin is described byBodansky et al. (13). Chloromethylated resins are commercially availablefrom Bio Rad Laboratories (Richmond, Calif.) and from Lab. Systems, Inc.The preparation of such a resin is described by Stewart et al. (8). BHAand MBHA resin supports are commercially available, and are generallyused when the desired polypeptide being synthesized has an unsubstitutedamide at the C-terminus. Thus, solid resin supports may be any of thoseknown in the art, such as one having the formulae --O--CH₂ -resinsupport, --NH BHA resin support, or --NH-MBHA resin support. When theunsubstituted amide is desired, use of a BHA or MBHA resin is preferred,because cleavage directly gives the amide. In case the N-methyl amide isdesired, it can be generated from an N-methyl BHA resin. Should othersubstituted amides be desired, the teaching of U.S. Pat. No. 4,569,967(14) can be used, or should still other groups than the free acid bedesired at the C-terminus, it may be preferable to synthesize thepeptide using classical methods as set forth in the Houben-Weyl text(7).

The C-terminal amino acid, protected by Boc and by a side-chainprotecting group, if appropriate, can be first coupled to achloromethylated resin according to the procedure set forth in K. Horikiet al. (15), using KF in DMF at about 60° C. for 24 hours with stirring,when a peptide having free acid at the C-terminus is to be synthesized.Following the coupling of the BOC-protected amino acid to the resinsupport, the α-amino protecting group is removed, as by usingtrifluoroacetic acid (TFA) in methylene chloride or TFA alone. Thedeprotection is carried out at a temperature between about 0° C. androom temperature. Other standard cleaving reagents, such as HCl indioxane, and conditions for removal of specific α-amino protectinggroups may be used as described in Schroder & Lubke (16).

After removal of the α-amino-protecting group, the remaining α-amino-and side chain-protected amino acids are coupled step-wise in thedesired order to obtain the intermediate compound defined hereinbefore,or as an alternative to adding each amino acid separately in thesynthesis, some of them may be coupled to one another prior to additionto the solid phase reactor. Selection of an appropriate coupling reagentis within the skill of the art. Particularly suitable as a couplingreagent is N,N'-dicyclohexylcarbodiimide (DCC).

The activating reagents used in the solid phase synthesis of thepeptides are well known in the peptide art. Examples of suitableactivating reagents are carbodiimides, such asN,N'-diisopropylcarbodiimide andN-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. Other activat- ingreagents and their use in peptide coupling are described by Schroder &Lubke (16) and Kapoor (17).

Each protected amino acid or amino acid sequence is introduced into thesolid-phase reactor in about a twofold or more excess, and the couplingmay be carried out in a medium of dimethylformamide (DMF): CH₂ Cl₂ (1:1)or in DMF or CH₂ Cl₂ alone. In cases where intermediate coupling occurs,the coupling procedure is repeated before removal of the α-aminoprotecting group prior to the coupling of the next amino acid. Thesuccess of the coupling reaction at each stage of the synthesis, ifperformed manually, is preferably monitored by the ninhydrin reaction,as described by Kaiser et al. (18). Coupling reactions can be performedautomatically, as on a Beckman 990 automatic synthesizer, using aprogram such as that reported in Rivier et al. (19).

After the desired amino acid sequence has been completed, theintermediate peptide can be removed from the resin support by treatmentwith a reagent, such as liquid hydrogen fluoride, which not only cleavesthe peptide from the resin but also cleaves all remaining side chainprotecting groups and also the α-amino protecting group at theN-terminus if it was not previously removed to obtain the peptide in theform of the free acid. If Met is present in the sequence, the Bocprotecting group is preferably first removed using trifluoroacetic acid(TFA)/ethanedithiol prior to cleaving the peptide from the resin with HFto eliminate potential S-alkylation. When using hydrogen fluoride forcleaving, one or more scavengers such as anisole, cresol, dimethylsufide and methylethyl sulfide are included in the reaction vessel.

Cyclization of the linear peptide is preferably affected, as opposed tocyclizing the peptide while a part of the peptidoresin, to create bondsbetween Cys residues. To effect such a disulfide cyclizing linkage,fully protected peptide can be cleaved from a hydroxymethylated resin ora chloromethylated resin support by ammonolysis, as is well known in theart, to yield the fully protected amide intermediate, which isthereafter suitably cyclized and deprotected. Alternatively,deprotection, as well as cleavage of the peptide from the above resinsor a benzhydrylamine (BHA) resin or a methylbenzhydrylamine (MBHA), cantake place at 0° C. with hydrofluoric acid (HF), followed by oxidationas described above.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1 Identification of δ-Conotoxin Peptide GmVIA Sequence

Specimens of C. gloriamaris were dissected, and the venom ducts wereremoved. The venom was squeezed and scraped from the ducts as describedby Cruz et al. (20) and then lyophilized. Tris-(2-carboxyethyl)phosphine(TCEP) was synthesized by the method of Burns et al. (21).

Lyophilized venom (200 mg) was first suspended in 5 mL of 0.1% TFA and40% acetonitrile and soaked for 10 minutes over ice with occasionalstirring and sonication. The solution was then centrifuged for 5 minutesusing a bench-top microfuge and the supernatant was saved. Theextraction procedure was repeated two more times with the same solventand twice with 5 mL of 0.1 TFA and 90% acetonitrile. Supernatants fromall extractions were combined and stored over ice while awaiting furtherpurification. The pellet was dried and weighed.

Pooled venom extracts were subjected to reversed-phase high pressureliquid chromatography (HPLC) using a C₈ Aquapore semipreparative column(7.0×250 mm; 4 mL/min). Secondary purification was carried out on a C₁₈Vydac column (218TP54, 4.6×250 mm; 1 mL/min). HPLC buffers were (A) 0.1%TFA in water, and (B) 0.09% TFA in 90% acetonitrile. For bothsemipreparative and analytical runs, peptides were eluted with a lineargradient of 1% buffer B increase/min. The C₁₈ Vydac analytical columnwas also used for purifying partially reduced intermediates and allkatedpeptides during disulfide bridge analysis.

Difulfides were reduced by incubating equal volumns of peptide solutionand 8 mM TCEP in 0.25 M Tris, pH 8.0, for 20 minutes at roomtemperature. A single product was obtained by reversed-phase HPLC on theC₁₈ Vydac analytical column. The reduced peptide was alkylated by theaddition of 1 μL of 4-VP/100 μL of peptide solution. After incubationfor 20-30 minutes in the dark, the solution was diluted to reduceacetonitrile concentration, and the pyridylethylated peptide wasrepurified by reversed-phse HPLC. The eluted peptide was adsorbed ontoBiobrene-treated glass fiber filters, and the amino acid sequence wasanalyzed by automated Edman degradation on an ABI Model 477A instrument.

Analysis of pyridylethylated peptide by standard Edman chemistry gavethe sequence shown in SEQ ID NO:1. Completeness of the sequence wasindicated by mass analysis using LSIMS. The observed (M+H)⁺ wasconsistent with the above sequence, having a free carboxyl at theC-terminus and three disulfide bridges (monoisotopic; observed (M+H)⁺=3352.5; theory, 3352.51). Thus, the peptide has 29 amino acids, and itssix cysteine residues are arrayed in the pattern (-C-C-CC-C-C-) typicalof the ω-conotoxins and δ-conotoxin TxVIA. Both δ-conotoxins lack theC-terminal amidation characteristic of most conotoxins.

Disulfide bridge analysis was carried out by the partial reductionmethod of Gray (6). δ-Conotoxin GmVIA in HPLC column effluent was addedto an equal volume of 20 nM TCEP in 0.17 M sodium citrate, pH 3.0, andincubated for 5 minutes at 64° C. The partially reduced species werepurified by HPLC and were immediately frozen in solution (pH 2.0) at-40° C. to prevent disulfide exchange. For convenience, and to minimizeloss of peptide, all intermediates were kept in the HPLC effluentwithout drying them down completely. Partially reduced peptides werealkylated by squirting 100 μL of thawed peptide solution into asupersaturated solution of IAM (100 mg in 200 μL of 0.5 M Tris-acetate,pH 8.0, containing 2 mM EDTA), while the latter was mixed rapidly. After20-30 seconds, the reaction was quenched by adding 450 μL of 0.5 Mcitric acid solution. Alkylated peptides were purified by HPLC(analytical C₁₈ Vydec column with flow rate of 1 mL/min and anacetonitrile gradient increase 1%/min) and were then subjected tocomplete reduction and further alkylation by 4-VP as describedpreviously.

The unusually high abundance of peptide in venom allowed the analyis ofits disulfide bridge connectivity, using partial reduction by TCAP at pH3.0 (6). No reduction was observed at room temperature, but a usefulspectrum of products was obtained after five minutes at 64° C.Completely reduced (R) and native (N) peptides, and four partiallyreduced intermediates (PR1-PR4) were seen. Peptides PR1, -2 and -3 werefurther purified and analyzed. Reaction with iosoacetamide, using therapid alkylation protocol proceeded with minimal rearrangement of thedisulfides. All remaining Cys residues were pyridylethylated followingfull reduction and alkylation with 4-VP. Thus, every cysteine residuewas converted either to Cys(CAM), if it had been part of a bridge whichwas reduced, or to Cys(PE), if it had been part of a bridge which hadinitially remained intact. The intermediates were then sequenced tolocate the two labels.

Analysis of PR2 revealed labeling of Cys4 and Cys19 with CAM, and of theremaining cysteines with PE. This indicates that a bridge linking Cys4and Cys19 was the only one which had been reduced. PR1 and PR3 proved tobe monocyclic peptides in which only Cys11-Cys24 and Cys18-Cys28remained intact, respectively. These results form a completelyconsistent set, indicating that the bridges are linked sequentially(4-19, 11-24 and 18-28) in the same pattern as that observed with theω-conotoxins.

For comparison, the disulfide bridge connectivities of the relatedpeptide ω-conotoxin TxVIA (27, 28) were also analyzed by the samemethods. A generally similar pattern was obtained after partialreduction and analysis of intermediates 2 and 3 was sufficient toestablish that the disulfide connectivity was the same as that ofδ-conotoxin GmVIA..

Example 2 Synthesis of δ-Conotoxin Peptide GmVIA Sequence

δ-Conotoxin GmVIA was synthesized by the two-stage strategy employed forω-conotoxin MVIID (22). The protected peptide resin was built usingstandard fmoc chemistry, couplings being carried out with equimolaramounts of amino acid derivative, DCC, HOBT. All amino acids werepurchased from Bahem (Torrance, Calif.), and side chains were protectedas follows: Arg (pmc), Asn (trt), Asp (t-bu), Gln (trt), Glu (t-bu), andLys (boc). Cys residues 4, 18, 19 and 28 were protected by trt, whileCys residues 11 and 24 were protected by acm.

At the completion of synthesis, the terminal fmoc group was removed bystandard treatment with piperidine (NMP (20% by volume. Peptide wasremoved from the resin by treatment for 2 hours at 20° C. with TFA/H₂O/ethanedithiol/phenol/thioanisole (90/5/2.5/7.5/5 by volume), and thewhole mixture was filtered rapidly into t-butyl methyl ether at -10° C.Linear peptide, retaining protection only on Cys11 and Cys24, wascollected as a pellet after centrifugation and was washed once witht-butyl methyl ether. The pellet dissolved readily in 60% acetonitrilecontaining 0.1% TFA; an ether layer that separated was discarded.Peptide solution was diluted with 0.1% aqueous TFA before application toa 2.5-×25-cm column of Vydac C₁₈. Elution was carried out at 20 mL/min,using a gradient of acetonitrile (27-45%) in 0.1% TFA. The majorpeptide-containing fraction was diluted with 30% acetonitrile, the pHwas adjusted to 7.0 with NaOH, and the solution was stirred overnight atroom temperature. This procedure oxidized Cys residues 4, 18, 19 and 28,generating three bicyclic isomers whih were isolated by HPLC. Insmall-scale trials, one of these gave native-like material afteroxidation with 2 mM I₂ in 10% TFA (10 min, 20° C., followed by a quenchwith 30 mM ascorbic acid). Oxidation was then carried out preparativelyon that isomer, and the tricyclic peptide was isolated by HPLC.

Construction of the protected peptide resin proceeded smoothly. A singlemajor product was obtained after deprotection and cleavage, acountingfor 53% of the total absorbance at 220 nm. Air oxidation of thismaterial gave the expected three bicyclic peptides, with a patternsimialr to that obtained for ω-conotoxin MVIID (22). Treatment of thesecond of these bicyclic peptides resulted in approximately 60%conversion to a product having the same elution time as natural peptide.Coinjection experiments verified that the two products wereindistinguishable by this method. Bioassays in the snail showed thatsynthetic peptide was equipotent with the natural.

Example 3 Identification of μO-Conotoxin Peptides MrVIA and MrVIB

Lyophilized crude venom (500 mg) was extracted (23), placed in aCentricon 30 microconcentrator, and centrifuged at 1500×g for 8 hours at4° C. Filtrate was purified on a C₁₈ Vydac column (22×250 mm) using agradient system. Buffer A=0.1% trifluoroacetic acid (TFA) and bufferB=0.1% TRA, 60% acetonitrile. The gradient began at 0% B, increased to15% B over 15 minutes, increased to 39% B over 72 minutes, increased to65% B over 15 minutes, increased to 100% B over five minutes, and heldat 100% B for 10 minutes. Flow rate was 10 ml/min. The very hydrophobicMrVIA and MrVIB elute as the last two major peaks at 109.4 and 110.3minutes. The fraction eluting at 109.4 minutes was subjected to a secondRPLC using a Vydac C₈ column (10×250 mm) with buffer A as above andbuffer B=0.1% TFA, 90% acetonitrile. The gradient began at 5% B,increased to 55% B over 15 minutes, and increased to 70% B over 45minutes. Flow rate was 5 ml/min. An analogous RPLC was performed toisolate MrVIB. Mass spectra were measured with a JEOL JMS-HX110double-focusing spectrometer fitted with a Cs⁺ gun. Sequencing wasperformed as previously described (24).

Liquid secondary ion mass spectrometry indicated that Cys residues arepresent as disulfides and that the C-terminal α-carboxyl group is thefree acid for both peptides (monoisotopic MH⁺ : MrVIA calculated3487.66, found 3487.8; MrVIB calculated 3404.58, found 3404.8).Biologically active μO-conotoxin MrVIA was synthesized in accordancewith the techniques previously described.

Example 4 Identification of DNA for μO-Conotoxin Peptide MrVIB

cDNA clones encoding the μO-conotoxin peptide MrVIB were isolated from asize-fractionated cDNA library constructed from C. magus venom ductmRNA. The cDNA library was size-fractionated into insets with averagesize of 74 Kb, 2 Kb, 1 Kb and 0.5 Kb. The two smallest size fractionswere screened.

Five μg of DNA were denatured in 0.4 M NaOH at 37° C. for 30 min. Thesolution was then neutralized by adding ammonium acetate to a finalconcentration of 0.4 M, and the DNA precipitated with two volumes ofabsolute ethanol. The DNA was pelleted, resuspended in 8 μl of H₂ O, andannealed with 2 pmols of primer by heating to 65° C. for 5 min andcooling slowly to 30° C. The DNA was sequenced using the SequenaseVersion 2.0 DNA sequencing kit. Labelling and termination reactions werecarried out according to protocol in the Sequenase Version 2.0 4thEdition Manual (United States Biochemical, 1990). Three cDNA clones forμO-conotoxin MrVIB were identified. The nucleic acid sequence andpresumptive translation product for the encoded precursor are shown asSEQ ID NO:5 and SEQ ID NO:6, respectively.

Example 5 Assay Methods

Bioassays and Biological Activities

Local garden snails, Helix aspersa, weighing 3-5 g, were chosen forbioassays because they were readily available. In the presence of food,the snails became aroused and no longer stayed in the shells. Once theywere fully active and out of the shells, the snails were put on ice toslow down their activity. Under these conditions the heads tend toremain extended, allowing easy injection of 10-140 μL aliquots of toxinfractions. The injection, similar to intracranial injection in thecommon mouse bioassay (25), was near the cerebral ganglion where nervesare heavily localized. For comparison, intracranial injection intotwo-week old mice was done with doeses of 20-60 nmol.

Electrophysiology

Isolated neurons from Aplysia californica or Aplysia aculifera werecultured as previously described (3, 26). The neurons were cultured atvery low densities to prevent synaptic interactions among them. Passiveand active membrane properties of the cultured neurons were studiesusing conventional intracellular recording and simulation techniques.Briefly, the cell body of a cultured neuron was impaled by amicroelectrode filled with 2 M KCl (4-10 MΩ resistance). The electrodewas used for both current injection and voltage recordings. Analysis ofthe resting potential, input resistance, and action potential aamplitudeand shape was carried out in artificial sea water (ASW) composed of 460mM NaCl, 10 mM KCl, 11 mM CaCl₂, 55 mM MgCl₂, an 10 mM HEPES, pH 7.6.The toxin for electrophysiological experiments was dissolved in ASWcontaining 10 mg/mL boving serum albumin and was applied to the bathingsolution tor each a final concentration of 0.3-0.75 μM.

Solutions

Control experiments were carried out in artificial sea water (ASW)composed of: 460 mM NaCl, 10 mM KCl, 11 mM CaCl₂, 55 mM MgCl₂ and 10 mMHEPES. The pH was adjusted to 7.6.

To minimize potassium currents, an external potassium channel blockingsolution (PCBS) was used, in which KCl was substituted for by CsCl. Inaddition, the solution contained 50 mM tetraethyl-ammonium chloride(TEA) and 0.1 mM 3,4-diaminopyridine (3, 4, DAP). The osmolarity of thesolution was adjusted by reducing the NaCl concentration to 410 mM.

To monitor sodium current, a calcium-free PCBS solution was used. Inthis solution, Ca²⁺ was substituted for by Mg²⁺, and the solution wassupplemented by 8 mM Co²⁺ to prevent any Ca²⁺ influx. For theseexperiments, the patch pipette contained: 440 CsCl, 40 CsGlutamate, 20NaCl, 2 MgCl₂, 10 Ethylenglycol-bis-(β-amino ethyl ether) N,N'-tetraacetic acid (BGTA), 100 N-[2-Hydroxyethyl]piperazine-N'[2-ethanesulfonic acid] (HEPES) and 3adenosine-5-triphosphate ATP) (The values are in mM). The pH wasadjusted to 7.3.

To monitor calcium currents, the external sodium ions were replaced byTEA. The patch pipette contained the same internal solution supplementedwith 0.5 mM guanosine-5'-triphosphate (GTP) to slow the run-down ofcalcium channels (31). The pH was adjusted to 7.3.

Potassium containing external and internal solutions were used whenoutward currents were under study. Sodium currents were eliminated byreplacing the sodium ions with Trisma 7.4 (Sigma, St. Louis). In theseexperiments, the patch pipette contained: 480 KCl, 20 NaCl, 2 MgCl₂, 10EGTA, and 100 HEPES (values are in mM). The pH was adjusted to 7.3.Throughout, the ± sign stands for standard deviation of the mean.

Competitive Binding with ω-Conotoxin GVIA

The procedures for membrane preparation and binding asay wereessentially as previously described by Cruz and Olivera (27), exceptthat NaCl was used instead of choline chloride in the wash medium of thebinding assay. Different concentrations of the peptide and ω-conotoxinGVIA were preincubated with the membrane preparation for 30 minutes onice before the addition of ¹²⁵ I-labelled GVIA.

Example 6 Biological Activity of δ-Conotoxin Peptide GmVIA

Because C. gloriamaris is believed to be a snail-hunting cone, theinitial in vivo bioassay used local garden snails. Volumes of peptidesolution between 10 and 40 μL were injected in the head region, near thecerebral ganglion. Injection of approximately 20 nmol of purifiedδ-conotoxin GmVIA induced retraction of the head and body into theshell; this was followed by secretion of viscous green slime and aconvulsive undulation into and out of the shell. Biological effects ongarden snails were deectable at a dose of 1.25 nmol/g and very obviousat 2 nmol/g. No apparent biological activity was observed when a muchgreater dose of peptide (10 nmol/g) was injected peritoneally into mice.

Electrophysiology

A preliminary study of the electrophysiological effects of purifiedtoxin was carried out on isolated Aplysia neurons. Changes in theresting potential, input resistance and action potential amplitude andshape upon addition of the toxin were assessed. The purified toxinrevealed significant effects at final concentrations of 0.3-0.75 μM.Within 10-60 seconds after bath application of the toxin, quiescentneurons fired spontaneously. Concomitantly, the action potentialduration increased by 1-2 orders of magnitude, extending in manyexperiments to over 250 ms. The changes in membrane excitability andaction potential duration induced by the toxin were completelyreversible upon washing of the neuron with ASW.

Toxin-induced prolongation of the action potential was still observedwhen K⁺ and Ca²⁺ conductances were blocked, suggesting that GmVIA'seffect is most likely due to a decrease in the rate of sodium currrentinactivation. For instance, in the experiment in which K⁺ conductanceswere blocked by using ASW in which KCl was replaced by CaCl. Inaddition, the solution contained 50 nM tetraethylammonium chloride and0.1 mM 3,4-diaminopyridine (osmolarity of the solution was restored byreducing the NaCl concentration to 410 mM). We refer to this solution aspotassium conductance blocking solution, or PCBS. Under theseconditions, bath application of 0.5 μM toxin prolonged the spikeduration. In the presence of the toxin and PCBS, bath application ofCa²⁺ to block Ca²⁺ current (final concentration of 8 mM) increased spikeduration even further. This is most likely due to blockage of residualCa²⁺ -dependent K⁺ conductances, which contribute to the repolarizationof the action potential.

These observations, together with preliminary whole-cell patch-clampstudies which were performed, indicate that the mechanism underlying thetoxin effect is a slowing down of sodium current inactivation, ratherthan changes in Ca²⁺ or K⁺ currents.

Action potential broadening by bath application of 0.4 μM δ-conotoxinGmVIA was observed. The altered action potential is composed of an earlypeak, followed by a long plateau of somewhat lower amplitude. Thisbroadening could be accounted for by several mechanisms, including thereduction of potassium conductances, an increase in the calcium orsodium conductances, or the activation of some latent calcium or sodiumvoltage gated channels.

To differentiate among these possibilities, experimental protocols thatpermitted examination of the isolated macroscopic currents of either K⁺,Ca²⁺ or Na⁺ were used.

Whether the toxin alters the potassium currents was first analyzed. Tothat end, the sodium and calcium currents were blocked as previouslydescribed. In one experiment, the neuron was depolarized for 250 ms froma holding potential of -50 mV to various values. Application ofδ-conotoxin GmVIA, at a final concentration of 0.5-2 μM, did not alterthe potassium current-voltage elations, nor its kinetics (n=4).

Whether the toxin alters calcium currents was next analyzed. For theseexperiments (n=4), the sodium and potassium currents were blocked aspreviously described; and as in (29). In an experiment, the neuron wasdepolarized from a holding potential of -50 mV to various potentials. Inthis experiment, as well as in others of the same kind, a reduction of10-20% in the amplitude of the calcium current was noticed during theexperiment (lasting 20-60 minutes), but the normalized current voltagerelationship of Ca²⁺ was not altered. The reduction in the peak calciumcurrent is most likely due to partial rundown of Ca²⁺ channels (31), asa similar gradual decrease in the calcium current was also observedduring the control periods. Thus, it appears that neither a reduction inpotassium currents nor an increase in the duration of the calciumcurrent can account for the effects of the toxin on spike shape.

To examine the effects of the toxin on the sodium current, a series ofexperiments (n=22) was performed in which the potassium currents andcalcium currents were eliminated as previously described.

The voltage clamp records (in an experiment in which the minimal currentwas evoked by a depolarizing voltage clamp step from a holding potentialof -50 mV to 22 mV) show that 0.5 μM δ-conotoxin GmVIA does not alterthe rise time of the sodium current but slows the rate of sodium currentinactivation. In the presence of the toxin, the sodium currentinactivation is composed of two phases, an early phase which is sloweddown in respect to the control and a second phase, which does not appearin control records altogether. This phase does not completely inactivateby the end of the voltage trace. In this and other experiments (n=8) inwhich Ca²⁺ and K⁺ currents were blocked, the decay of the sodium currentin the control experiments was exponential, with a single time constantτ=0.35 ms (average τ=0.47±0.14 ms, n=8). Following the application ofδ-conotoxin GmVIA, the rate of sodium current inactivation decreased andwas clearly composed of two phases. The semi-log plot of theinactivation phase shows that from about 10 ms onwards, the inactivationtime constant was 359.6 ms (average τ=488±120 ms, n=8). The inactivationrate of the early phase, obtained by subtracting the extrapolated slopefrom the slope of the earlier phase of the codium currnt, increased from0.35 ms in control to 0.84 ms (average of 0.86±0.12 ms, n=8) afterapplication of δ-conotoxin GmVIA. While the inactivation rate of thesodium current was altered, the current voltage relationship of theearly sodium current was not changed.

δ-Conotoxin GmVIA (0.5 μM) changed the steady-state voltage inactivationof the sodium channels. For this experiment, the holding potential(V_(h)) was set at various values ranging from -90 to 0 in V for 2seconds. Sodium currents were generated by stepping the voltage to 22 mVfor 15 minutes in Ca²⁺ -free PCBS and 8 mM Co²⁺. The peak of earlysodium conductance (G_(Na+)) was plotted as a function of the maximalsodium conductance (G_(Na+) /G_(Na+) max) observed when V_(h) was -50mV. The solid line shows: G_(Na+) /G_(Na+) max ={1+exp [(V_(h)-V₀.5))/S]}⁻¹, where V₀.5 is the half-inactivating voltage, and S is theslope parameter fitting the experimental data. Following bathapplication of the toxin, the inactivation curve shifted to morepositive potentials and V₀.5 was -21 mV, compared with the value incontrol in which V₀.5 =30 mV. The slopes of the inactivation curve incontrol and δ-conotoxin GmVIA treated neuron were the same (slopeparameter=4.6).

It is interesting that steady state voltage inactivation of the slowinactivating phase of sodium current behaves precisely as the earlyphase of sodium current.

Activation of the early and late sodium current as a fraction of themaximal sodium conductance was analyzed. To determine the inactivationof the early sodium current in control experiments and in the presenceof δ-conotoxin GmVIA, the membrane potential was set to -50 mV and thenclamped for 0.5 minutes to various potentials ranging from -40 to 60 mV,in 5 mV increments. The resulting tail currents were measured andexpressed as relative conductances. The activation curve of the slowlyinactivating current was determined only in the presence of the toxin.This was done by setting the holding voltage to -50 mV and then clampingthe membrane potential to various values (from -40 to 60 mV) for aduration of 15 minutes. The resulting tail current under theseconditions consisted only of the slowly-inactivating phase, since theearly sodium current was inactivated. The peak of the early sodiumconductance and the peak of the late slowly inactivating conductance(G_(Na+)) were plotted as a function of the maximal sodium conductanceG_(Na+) /G_(Na+max)) observed when V_(m) was mV. The solid line shows:G_(Na+) /G_(Na+max) ={1=exp [-(V_(m) -V₀.5)/S]}⁻¹, where V_(m) is thetesting potential, V0.5 is the half-activating voltage and S is theslope parameter fitting the experimental data. Following bathapplication of 0.4-0.7 μM toxin, the activation curve of the earlysodium current is shifted to more negative potentials, and V₀.5 was 10mV compared to the value in control where V₀.5 =15 mV. The V₀.5 of latesodium current is 4 mV. The slopes of the activation curves in controland δ-conotoxin GmVIA treated neurons (the early sodium current and thelate sodium current) are the same (slope parameter=4.75).

The relative refractory period of the early and the slowly inactivatingphases of the sodium currents were studied by delivering two consecutivedepolarizing voltage clamp pulses to the neuron. The first depolarizingpulse lasted for 10 minutes and the second for 25 minutes, the timeinterval between the two pulses was gradually reduced, and the sodiumcurrents were monitored in control and following toxin application.Prior to application of the toxin (0.5 μM), both pulses evoked the earlyinactivating sodium currents. The amplitude of the sodium currentinduced by the second pulse decreased as the time interval between thetwo pulses was reduced. Toxin application induced the appearance of theslowly inactivating phases of the sodium current not seen in thecontrol. The amplitude of the slowly inactivating sodium current wasalmost constant independent of the time interval between the first andsecond voltage clamp pulses. It was also interesting to note that theamplitude of the slowly inactivating sodium current is independent ofthe amplitude of the early sodium current.

Comparison Between Effects of δ-Conotoxin GmVIA and δ-Conotoxin TxVIA

The electrophysiological effects of δ-conotoxin GmVIA and TxVIA isolatedfrom another mollusc hunting-snail Conus textile on cultured Aplysianeurons (3, 32) were quite similar. Both toxins induced action potentialbroadening and increased excitability by slowing the rate of sodiumcurrent inactivationwith no significant effects on either the rise timeor the peak of the voltage-activated sodium current. However, there weredifferences between the efects of these two toxins. For this experiment(n=5), potassium and calcium currents were eliminated and the neuron wasclamped from a holding potential of -50 mV to 22 mV. The effects of thetwo peptides were studied sequentially on the same neuron. First, theneuron was exposed to 0.5 μM GmVIA, then thoroughly washed until thesodium current recovered to control levels. TxVIA was then applied andinduced a prolongation of the sodium current. The superimposed tracesclearly demonstrated that δ-conotoxin GmVIA induced a much longer slowlyinactivating phase of the sodium current than δ-conotoxin TxVIA. Similarobservations were made when the order of toxin application was reversed.The differences in the inactivation kinetics of the sodium current arenot due to differences in the affinities of the two toxins for thesodium channels, since exposure of the neurons to higher concentrationsof δ-conotoxin TxVIA never altered the duration of the slowlyinactivating current to the same extent as did δ-conotoxin GmVIA.

Competitive Binding with ω-Conotoxin GVIA

At concentrations up to 5.0 μM of the test peptide, δ-conotoxin GmVIAdid not compete with ¹³⁵ I-labelled ω-conotoxin GVIA on brain membranepreparations from frogs, chicks and rats. Positive controls withunlabelled ω-conotoxin GVIA gave the expected level of competition: 25nM unlabelled toxin displaced approximately 90% of ¹²⁵ I-GVIA, and 250nM competed out approximately 98% of label.

Discussion

δ-Conotoxin GmVIA, the major component present in the venom of themollusc hunting snail Conus gloriamaris, elicits spike broadening atconcentration of 5 μM. Voltage clamp analysis reveals that the effectsof the toxin are expressed by: Slowing down the rate of the early sodiumcurrent inactivation; induction of a late slowly inactivating sodiumcurrent which is not detectable in control experiments; Shifting thesteady state sodium current inactivation curve to more depolarizedvalues and shifting the activation curve to more hyperpolarized values.These changes are not associated with an increase in the rate of rise ofthe early sodium current or by a change in the peak sodium current.

The experiments show that the toxin modifies the sodium currentinactivation kinetics from a single exponential with an averageτ=0.47±0.14 minutes to a slower decay, which can be described by twotime constants: the initial inactivation phase has a τ=0.86±0.12 minuteand the second phase a τ=488±120 minutes. These changes in timeconstants may account for the typical alterations in the actionpotential shape induced by δ-conotoxin GmVIA. In ASW and in the presenceof the toxin, the early fast phase of the action potential and itsplateau corresponds to the early rapidly inactivating sodium current andthe long-lasting slowly inactivating phase.

The amplitude of the late phase of the sodium current induced byδ-conotoxin GmVIA is independent of the amplitude of the early current.Thus, even when the early current is completely inactivated by aprepulse, the amplitude of the late component is not reduced. Thesimplest explanation to account for this observation is that the toxinalters the inactivation kinetics of the entire population of sodiumchannels from a single time constant to dual time constants. If thisassumption is right, then the observations suggest that in the presenceof δ-conotoxin-GmVIA, the sodium channels can be activated in two modes:In the one mode they go through the rapid inactivation phase to the slowinactivating phase, or alternatively are "switched" directly into theslowly inactivating mode without first going through the rapidlyinactivating phase.. An alternative explanation assumes that the toxinactivates a population of silent, slowly inactivating sodium channels.This hypothesis is also consistent with the finding tht thenon-inactivating phase can be activated independently of the earlysodium current. However, since the peak of the early sodium current isnot increased in the presence of the toxin as would be expected ifadditional sodium channels were activated, then this hypothesis seems tobe less likely.

The macroscopic effects of the toxin purified from the venom of theshallow water molusc hunting snail C. textile (3) and the one purifiedfrom the deep water C. gloriamaris are similar but not identical. Bothtoxins alter the inactivation kinetics of the sodium current from aprocess that is best described by a single exponent into a biphasicprocess. However, the effects of the two toxins differ becauseδ-conotoxin GmVIA induces a significantly longer, slow inactivationphase than that induced by δ-conotoxin TxVIA. Additionally, the effectsof δ-conotoxin TxVIA undergo desensitization in the presence of thetoxin, whereas the effects of δ-conotoxin GmVIA are observed for as longas the toxin is present in the bathing solution. It is reasonable toassume that these peptide toxins target the same ligand binding pocketof the sodium channel: Both peptides are extremely hydrophobic, acharacteristic which may be important for binding to this specific site.Nevertheless, the differences in the primary sequences of these peptidescause a clear difference in their detailed effects on sodium currentinactivation kinetics.

Example 7 Biological Activity of μO-Conotoxin Peptide MrVIA

Blockade of Action Potential and Inward Sodium Current by μO-ConotoxinMrVIA as Revealed by Current and Voltage Clamp Experiments

The current clamp experiments were carried out by a microelectrodeinserted into the cell body of a cultured Aplysia neuron. The electrodewas used for both current injection and voltage recording. To minimizethe potassium conductances, the current clamp experiments were carriedout in artificial sea water containing 50 mM tetraethylammonium chloride(TEA) and 0.3 mM 3,4-diainopyridine (3,4-DAP)(3). (A₁) control: Theaction potential was generated by an intracellular rectangulardepolarizing pulse. (A₂): Ten seconds after toxin application to reach afinal bath concentration of 350 nM, the action potential was blocked. Anincrease in the stimulus intensity after the blockade of the actionpotential failed to elicit a regenerative response. To directly examinethe toxin action on sodium, calcium and potassium currents, thewhole-cell patch clamp configuration was used. Adequate space clamp wasachieved by trimming off the main axon of the neuron prior to theexperiment (33, 34). To monitor only the sodium current (I_(Na+)), thepatch clamp eperiments were carried out in an external solution composedof: 410 NaCl, 10 CsCl, 66 MgCl₂, 9 CoCl₂, 50 TEA, 0.3 3,4-DAP. The pathelectrode contained 440 CsCl, 40 CsGlutamate, 20 NaCl, 2 MgCl2, 10 EGTA,100 HEPES and 3 ATP (the values are given in mM). The inward I_(Na+)evoked by depolarizing the neuron from a holding potential of -50 to 20mV was completely blocked 30 seconds following the application of 250 nMMrVIA. Partial blockage of I_(Na+) by 40 nM toxin revealed that theblock is not associated with a change in the current voltagerelationships. Patch clamp experiments revealed that calcium andpotassium currents are not affected by the toxin.

The μO precursor sequence can be readily aligned with the precursorsequences of ω-conotoxin GVIA (36) as well as with δ-conotoxin TxVIAfrom Conus textile (37). Extensive sequence identity between the μO-, ω-and δ-conotoxin precursors is observed. Although structurally related,the peptides are functionally divergent. Binding competition experimentsdemonstrate that competitive binding inhibition by μO-conotoxin MrVIAdoes not occur for the high affinity ω-conotoxin GVIA binding site onmammalian brain Ca²⁺ channels. Electrophysiological experiments showthat μO-conotoxin MrVIA and δ-conotoxin TxVIA elicit oposite effects,since δ-conotoxin TxVIA is an excitotoxin which increases Na⁺conductance (3). Thus, although μO-, ω- and δ-onotoxins apparentlybelong to the same protein superfamily, they have strickingly differentphysiological effects. In contrast, the functionally homologousμ-conotoxin GIIIA has an unrelated disulfide structure, and itsprecursor sequence shows no homology whatsoever to the μO-conotoxinsfrom C. marmoreus. Thus, the peptides provide molecular guideposts forspecies diversification in this genus. The genetic analysis shows thatthe μO-conotoxins, Na channel inhibitors from C. marmoreus, wereindependently evolved from the μ-conotoxins from fish-hunting Conus.Thus, in this single genus, one protein superfamily comprises multiplefunctionally-distinct toxin clases, but functional convergence of twosodium channel-blocking toxins from different superfamilies is alsoobserved.

In Conus textile venom one peptide, δ-conotoxin TxVIA, is present athigher levels than any other (27). The purified δ-conotoxin induces theconvulsive contractures in snails observed with whole C. textile venom.In contrast, μO-conotoxin MrVIA causes the flaccid relaxationcharacteristic of crude C. marmoreus venom. Thus, the two peptides, bothmajor components of their respective venoms, are likely to play keyroles in the contrasting physiological strategy that these twosnail-hunting Conus adopt to cause immobilization outside the shell. Thetwo peptides target the same macromolecular complex (thevoltage-sensitive sodium channel) but Conus textile increasesexcitability by inhibiting channel inactivation through its δ-conotoxinwhile Conus marmoreus decreases excitability by blocking channelconductance via its μO-conotoxin.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

LIST OF REFERENCES

1. Myers, R. A. et al. (1993). Conus Peptides as Chemical Probes forReceptors and Ion Channels. Chem.Rev. 93:1923-1936.

2. Miljanich, G. P. et al. (1993). U.S. Pat. No. 5,264,371.

3. Hasson, A. et al. (1993). Eur. J. Neurosci. 5:56-64.

4. Olivera, B. M. et al. (1984). U.S. Pat. No. 4,447,356.

5. Sambrook, J. et al. (1979). Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

6. Gray, W. R. (1993). Disulfide Structures of Highly Bridged Peptides:A New Strategy for Analysis. Protein Science 2:1732-1748.

7. "Methoden der Organischen Chemie (Houben-Weyl): Synthese vonPeptiden," E. Wunsch (Ed.), Georg Thieme Verlag, Stuttgart, Ger. (1974).

8. Stewart and Young, Solid-Phase Peptide Synthesis, Freeman & Co., SanFrancisco, Calif. (1969).

9. Vale et al. (1978). U.S. Pat. No. 4,105,603.

10. U.S. Pat. No. 3,972,859 (1976).

11. U.S. Pat. No. 3,842,067 (1974).

12. U.S. Pat. No. 3,862,925 (1975).

13. Bodansky et al. (1966). Chem. Ind. 38:1597-98.

14. U.S. Pat. No. 4,569,967.

15. Horiki, K. et al. (1978). Chemistry Letters 165-68.

16. Schroder & Lubke (1965). The Peptides 1:72-75, Academic Press, N.Y.

17. Kapoor (1970). J. Pharm. Sci. 59:1-27.

18. Kaiser et al. (1970). Anal. Biochem. 34:595.

19. Rivier J. R. et al. (1978). Biopolymers 17:1927-38.

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LIST OF REFERENCES (Cont'd)

21. Burns, J. A. et al. (1991). Selective reduction of disulfides bytris-[2-carboxyethyl]phosphine, J. Org. Chem. 56:2648-50.

22. Monje, V. D. et al. (1993). Neuropharm. 32:1149.

23. McIntosh, J. M. et al. (1994). J.Biol. Chem. 269:16733-16739.

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32. Spira, M. E. et al. (1993). Isr. J. Med. Sci. 29:530-543.

33. Hamill, O. P. et al. (1981). Improved patch clamp techniques forhigh-resolution current reading from cells and cell-free membranepatches. Plfgers Arch. 391:85-100.

34. Benbassat, D. and Spira, M. E. (1993). J. Exp. Neurol. 122:295-300.

35. Spira, M. E. et al. (1993). J. Neurobiol. 24:300-316.

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37. Woodward, S. R., et al. (1990). EMBO J.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 6                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 29 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Conus glo - #riamaris                                           (B) STRAIN: GmVIA                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 4..19                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 11..24                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 18..28                                                #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Val Lys Pro Cys Arg Lys Glu Gly - # Gln Leu Cys Asp Pro Ile Phe        Gln                                                                           #   15                                                                        -      Asn Cys Cys Arg Gly Trp Asn Cys - # Val Leu Phe Cys Val                #                 25                                                          - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 31 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Conus mar - #moreus                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/note= "Xaa at residue 3 is Arg or                                                          Ser"                                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/note= "Xaa at residue 14 is Ile or                                                         Leu"                                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                    #/note= "Xaa at residue 17 is Ile or                                                         Val"                                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 2..20                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 9..25                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 19..30                                                #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ala Cys Xaa Lys Lys Trp Glu Tyr - # Cys Ile Val Pro Ile Xaa Gly        Phe                                                                           #   15                                                                        -      Xaa Tyr Cys Cys Pro Gly Leu Ile - # Cys Gly Pro Phe Val Cys Val        #                 30                                                          - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 31 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Conus mar - #moreus                                             (B) STRAIN: MrVIA                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 2..20                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 9..25                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 19..30                                                #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ala Cys Arg Lys Lys Trp Glu Tyr - # Cys Ile Val Pro Ile Ile Gly        Phe                                                                           #   15                                                                        -      Ile Tyr Cys Cys Pro Gly Leu Ile - # Cys Gly Pro Phe Val Cys Val        #                 30                                                          - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 31 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Conus mar - #moreus                                             (B) STRAIN: MrVIB                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 2..20                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 9..25                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 19..30                                                #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ                                      -      Ala Cys Ser Lys Lys Trp Glu Tyr - # Cys Ile Val Pro Ile Leu Gly        Phe                                                                           #   15                                                                        -      Val Tyr Cys Cys Pro Gly Leu Ile - # Cys Gly Pro Phe Val Cys Val        #                 30                                                          - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 255 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Conus mar - #moreus                                             (B) STRAIN: MrVIB                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..246                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 1..66                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 154..246                                              #ID NO:5: (xi) SEQUENCE DESCRIPTION: SEQ                                      - ATG AAA CTG ACG TGC ATG ATG ATC GTT GCT GT - #G CTG TTC TTG ACA GCC           48                                                                          Met Lys Leu Thr Cys Met Met Ile Val Ala Va - #l Leu Phe Leu Thr Ala           40                                                                            - TGG ACG CTC GTC ATG GCT GAT GAC TCC AAC AA - #T GGA CTG GCG AAT CAT           96                                                                          Trp Thr Leu Val Met Ala Asp Asp Ser Asn As - #n Gly Leu Ala Asn His           - #20                                                                         - TTT TTG AAA TCA CGT GAC GAA ATG GAG GAC CC - #C GAA GCT TCT AAA TTG          144                                                                          Phe Leu Lys Ser Arg Asp Glu Met Glu Asp Pr - #o Glu Ala Ser Lys Leu           - GAG AAA AGG GCG TGC AGC AAA AAA TGG GAA TA - #T TGT ATA GTA CCG ATC          192                                                                          Glu Lys Arg Ala Cys Ser Lys Lys Trp Glu Ty - #r Cys Ile Val Pro Ile           #           10                                                                - CTT GGA TTC GTA TAT TGC TGC CCT GGC TTA AT - #C TGT GGT CCT TTC GTC          240                                                                          Leu Gly Phe Val Tyr Cys Cys Pro Gly Leu Il - #e Cys Gly Pro Phe Val           #     25                                                                      #   255                                                                       Cys Val                                                                        30                                                                           - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 82 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:6:                       - Met Lys Leu Thr Cys Met Met Ile Val Ala Va - #l Leu Phe Leu Thr Ala         40                                                                            - Trp Thr Leu Val Met Ala Asp Asp Ser Asn As - #n Gly Leu Ala Asn His         - #20                                                                         - Phe Leu Lys Ser Arg Asp Glu Met Glu Asp Pr - #o Glu Ala Ser Lys Leu         5                                                                             - Glu Lys Arg Ala Cys Ser Lys Lys Trp Glu Ty - #r Cys Ile Val Pro Ile         #           10                                                                - Leu Gly Phe Val Tyr Cys Cys Pro Gly Leu Il - #e Cys Gly Pro Phe Val         #     25                                                                      - Cys Val                                                                      30                                                                           __________________________________________________________________________

What is claimed is:
 1. An isolated DNA coding for a conotoxin MrVIBpolypeptide having the amino acid sequence set forth in SEQ ID NO:6. 2.The isolated DNA of claim 1, wherein said DNA comprises the nucleotidesequence set forth in SEQ ID NO:5.